TGF-? superfamily members play a central role in regulation of hypertrophic, inflammatory, and fibrotic responses in failing and remodeling hearts, modulating phenotype and function of both cardiomyocytes and interstitial cells. TGF-?s act by stimulating a series of intracellular effectors the receptor-activated Smads (R-Smads), or through Smad-independent pathways. Endogenous negative regulators of TGF-? signaling cascades may play an important protective role in cardiac remodeling, by restraining fibrotic or hypertrophic responses. The inhibitory Smads (I-Smads), Smad6 and Smad7 have been implicated in negative regulation of TGF-? responses in many cell types. The current proposal uses newly-generated cell-specific knockout mice to investigate for the first time the role of the I-Smads, Smad6 and Smad7 in regulation of cardiac remodeling in the pressure-overloaded heart. Our preliminary data demonstrate induction of Smad6 and Smad7 in cardiomyocytes, fibroblasts and macrophages, but not in lymphocytes and neutrophils infiltrating the pressure-overloaded myocardium, and suggest critical roles of cardiomyocyte and fibroblast-specific Smad7 in protection of the heart from adverse remodeling and dysfunction. The role of the cell- specific actions of the I-Smads and the molecular signals modulated by Smad6 and Smad7 will be explored in 3 specific aims: Specific aim 1: to explore the role of Smad7 in regulation of cardiomyocyte, fibroblast and macrophage phenotype in the pressure-overloaded heart. Our preliminary studies show that Smad7 is markedly upregulated following cardiac pressure overload, and is localized in cardiomyocytes, activated myofibroblasts, and macrophages, but not in lymphocytes and neutrophils. Accordingly, we will study cell-specific mechanisms of Smad7 regulation, and we will use cardiomyocyte, fibroblast/myofibroblast, and myeloid cell-specific Smad7 knockout mice, recently generated by our laboratory, to explore the cellular effects of Smad7 in the pressure-overloaded myocardium. Specific aim 2: to dissect the molecular mechanisms responsible for the effects of Smad7 in vivo and in vitro. Smad7 actions may involve modulation of R-Smad-dependent pathways, effects on Smad-independent signaling cascades, or interactions with TGF-?-independent signals. The molecular mechanisms for Smad7-dependent regulation of cardiomyocyte, fibroblast and macrophage phenotype, and the paracrine signals involved in regulation of fibrogenic, inflammatory and hypertrophic responses, will be studied in vitro and in vivo, using both loss and gain-of-function approaches. Specific aim 3: to investigate the role of Smad6 in remodeling of the pressure-overloaded myocardium. Our preliminary studies show induction of Smad6 in the pressure-overloaded myocardium, and localization in cardiomyocytes, fibroblasts and macrophages. Conditional Smad6 knockout mice will be used to dissect the cell-specific actions of Smad6 in the pressure-overloaded heart, and the mechanisms responsible for Smad6-mediated effects will be explored in vivo and in vitro. The proposal investigates for the first time the role of Smad6 and Smad7 in cardiac remodeling, dissecting their molecular targets and mechanisms of action. The significance of the proposed experiments extends beyond the cardiovascular field, providing new insights into the biology of the TGF-? superfamily.